A diverse collection of protein-nucleic acid complexes and membrane-bound structures are released from mammalian cells during the course of their life and death (FIG. 1). Such compositions are broadly termed “bioparticles”. Exemplary protein-nucleic acid complexes include Ago2-microRNA complexes, which are known to exist as stable complexes in cell-free biofluids (Arroyo et al. Argonaute2 Complexes Carry a Population of Circulating MicroRNAs Independent of Vesicles in Human Plasma (2011) PNAS 108:5003-5008). Such complexes are released into the fluids of a subject (e.g., urine, blood, etc.) according to the status of the cell and/or upon degradation of the cell after death.
Membrane-bound structures (also known as extracellular vesicles or microvesicles) released from or otherwise derived from cells include exosomes, microvesicles, apoptotic bodies, and high density lipoprotein (HDL)-particles. (It is noted that the terms “extracellular vesicles” and “microvesicles” are used interchangeably herein to describe all cell-derived membrane-bound structures.)
The function of extracellular vesicles is not clearly understood, although they are theorized to act as nano-shuttles for the transport and delivery of information from one location and/or cell type to distant locations and/or other cell types (Mathivanan and Simpson, “Exosomes: extracellular organelles important in intercellular communication,” J. Proteomics 73(10):1907-1920 (2010)). Also, they are theorized to be involved in a wide variety of physiological processes, including cardiac disease, adaptive immune responses to pathogens, and in tumor biology. It is suggested that microvesicles may play roles in tumor immune suppression, metastasis, and tumor-stroma interactions. Microvesicles are thought to play a role in immune system cellular communication, for example, involving dendritic cells and B cells (Raposo et al., J. Exp. Med. 183 (1996) 1161).
The ubiquitous presence of circulating microvesicles in body fluids, their association with a broad range of physiological processes, as well as their elevated levels in human disease, suggest that microvesicles can potentially serve as tools in molecular medicine as measures of physiological state, disease diagnostics, and possibly therapeutic targeting.
Although the study of microvesicles/exosomes had been greatly advanced with the development of analytical systems such as nanoparticle tracking analysis (NTA) and fluorescent nanoparticle tracking analysis (FNTA; see (i) Van der Pol et al., “Optical and non-optical methods for detection and characterization of microparticles and exosomes,” Journal of Thrombosis and Haemostasis (2010), doi: 10.1111/j.1538-7836.2010.04074.x; (ii) Dragovic et al., “Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis,” Nanomedicine: Nanotechnology, Biology and Medicine (2011), doi:10.1016/j.nano.2011.04.003, other technical challenges remain.
One of the significant technical challenges in current research in microvesicles is the problem of how to efficiently isolate the microvesicles from various sources. Current methodologies to isolate secreted microvesicles, including but not limited to exosomes, are constrained by technical limitations and other drawbacks. These known methodologies are labor intensive, time-consuming, costly, and can be unreliable for different fluids; see Tauro et al., “Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes,” Methods 56(2):293-304 (print February 2012, Epub Jan. 21, 2012), doi:10.1016/j.ymeth.2012.01.002.
Historically, ultracentrifugation is the traditional method for microvesicle isolation. Generally, centrifugation is the process whereby a centrifugal force is applied to a mixture, whereby more-dense components of the mixture migrate away from the axis of the centrifuge relative to other less-dense components in the mixture. The force that is applied to the mixture is a function of the speed of the centrifuge rotor, and the radius of the spin. In most applications, the force of the spin will result in a precipitate (a pellet) to gather at the bottom of the centrifuge tube, where the remaining solution is properly called a “supernate” or “supernatant.” In other similar applications, a density-based separation or “gradient centrifugation” technique is used to isolate a particular species from a mixture that contains components that are both more dense and less dense than the desired component (e.g., OptiPrep™).
During the circular motion of a centrifuge rotor, the force that is applied is the product of the radius and the angular velocity of the spin, where the force is traditionally expressed as acceleration relative to “g,” the standard acceleration due to gravity at the Earth's surface. The centrifugal force that is applied is termed the “relative centrifugal force” (RCF), and is expressed in multiples of “g” (or “×g”).
The centrifugation procedures that have been used to isolate circulating microvesicles can incorporate as many as five centrifugation steps, with at least two of these spins requiring centrifugal forces in excess of 100,000×g for several hours. Generally, ultracentrifugation is centrifugation conditions that produce forces in excess of 100,000×g. These ultracentrifugation procedures are time consuming and labor intensive, and furthermore, are constrained by the requirement for expensive ultracentrifugation equipment. They can also be unreliable for certain fluids (see FIGS. 2 and 3).
Size exclusion chromatography can also be used to isolate microvesicles, for example, by using a Sephadex™ G200 column matrix. This approach is also time consuming and the yields are inconsistent. It also may be difficult or expensive to scale up to larger quantities of biofluid. Finally, these columns can be clogged by viscous biofluids.
Selective immunoaffinity capture (including immuno-precipitation) can also be used to isolate circulating microvesicles, for example, by using antibodies directed against the epithelial cell adhesion molecule, a type-1 transmembrane cell-surface protein (also known as EpCAM, CD326, KSA, TROP1). The anti-EpCAM antibodies can be coupled to magnetic microbeads, such as Dynabeads® magnetic beads. This method has very low yields compared to other methods, and is costly due to the use of the immuno-reagents and magnetic beads, and further, these system reagents cannot be reused for subsequent isolations.
What is needed in the art are methods for the rapid and inexpensive isolation of extracellular membrane particles, including microvesicles, exosomes, and apoptotic bodies, as well as any accompanying biomarkers, especially from biofluids such as urine. It would also be useful to have such a method that would isolate membrane-free protein-nucleic acid particles, cell-free messenger RNA, and cell-free DNA as well. Finally, for many applications, it would be desirable to obtain intact bioparticles for use in mechanistic, vaccine-related, delivery-related and therapeutic studies.
Such methods will ideally use common laboratory reagents and apparatus, and will not require high-speed centrifugation, such as ultracentrifugation. In addition, methods that provide higher yields than current methods are also needed, allowing for the isolation of important biomarkers and/or therapeutic targets from a smaller volume of sample.
Furthermore, what is also needed in the art are methods for generating cell culture media that are free of endogenous bioparticles, or have reduced concentrations of endogenous bioparticles compared to traditional complete media.